This invention relates to devices and methods for performing active, multi-step molecular and biological sample preparation and diagnostic analyses. More particularly, the invention relates to sample preparation, cell selection, biological sample purification, complexity reduction, biological diagnostics and general sample preparation and handling.
Molecular biology comprises a wide variety of techniques for the analysis of nucleic acid and protein. Many of these techniques and procedures form the basis of clinical diagnostic assays and tests. These techniques include nucleic acid hybridization analysis, restriction enzyme analysis, genetic sequence analysis, and the separation and purification of nucleic acids and proteins (See, e.g., J. Sambrook, E. F. Fritsch, and T. Maniatis, Molecular Cloning: A Laboratory Manual, 2 Ed., Cold spring Harbor Laboratory Press, Cold Spring Harbor, N.Y., 1989).
Most of these techniques involve carrying out numerous operations (e.g., pipetting, centrifugations, electrophoresis) on a large number of samples. They are often complex and time consuming, and generally require a high degree of accuracy. Many a technique is limited in its application by a lack of sensitivity, specificity or reproducibility. For example, these problems have limited many diagnostic applications of nucleic acid hybridization analysis.
The complete process for carrying out a DNA hybridization analysis for a genetic or infectious disease is very involved. Broadly speaking, the complete process may be divided into a number of steps and substeps. In the case of genetic disease diagnosis, the first step involves obtaining the sample (e.g., blood or tissue). Depending on the type of sample, various pre-treatments would be carried out. The second step involves disrupting or lysing the cells, which then releases the crude DNA and RNA (for simplicity, a reference to DNA in the following text also refers to RNA, where appropriate) material along with other cellular constituents. Generally, several sub-steps are necessary to remove cell debris and to purify further the crude lysate. At this point several options exist for further processing and analysis. One option involves denaturing the purified sample DNA and carrying out a direct hybridization analysis in one of many formats (dot blot, microbead, microtiter plate, etc.). A second option, called Southern blot hybridization, involves cleaving DNA with restriction enzymes, separating the DNA fragments on an electrophoretic gel, blotting to a membrane filter, and then hybridizing the blot with specific DNA probe sequences. This procedure effectively reduces the complexity of the genomic DNA sample, and thereby helps to improve the hybridization specificity and sensitivity. Unfortunately, this procedure is long and arduous. A third option is to carry out the polymerase chain reaction (PCR) or other amplification procedure. The PCR procedure amplifies (increases) the number of target DNA sequences. Amplification of target DNA helps to overcome problems related to complexity and sensitivity in analysis of genomic DNA or RNA. All these procedures are time consuming, relatively complicated, and add significantly to the cost of a diagnostic test. After these sample preparation and DNA processing steps, the actual hybridization reaction is performed. Finally, detection and data analysis convert the hybridization event into an analytical result.
The steps of sample preparation and processing have typically been performed separate and apart from the other main steps of hybridization and detection and analysis. Indeed, the various substeps comprising sample preparation and DNA processing have often been performed as a discrete operation separate and apart from the other substeps. Considering these substeps in more detail, samples have been obtained through any number of means, such as obtaining of whole blood, tissue, or other biological fluid samples. In the case of blood, the sample is often processed to remove red blood cells and retain the desired nucleated (white) cells. This process is usually carried out by density gradient centrifugation. Cell disruption or lysis is then carried out, preferably by the technique of sonication, freeze/thawing, or by addition of lysing reagents.
In certain cases, the blood is extensively processed to remove contaminants. One such system known to the prior art is the Qiagen system. This system involves prior lysis followed by digestion with proteinase K, after which the sample is loaded onto a column and then eluted with a high salt buffer (e.g., 1.25 M NaCl). The sample is concentrated by precipitation with isopropanol and then centrifuged to form a pellet. The pellet is then washed with ethanol and centrifuged, after which it is placed in a desired buffer. The total purification time is greater than approximately two hours and the manufacturer claims an optical density ratio (260 nm/280 nm) of 1.7 to 1.9 (OD 260-280). The high salt concentration can preclude performance of certain enzymatic reactions on the prepared materials. Further, DNA prepared by the Qiagen method has relatively poor transport on an electrophoretic diagnostic system using free field electrophoresis.
Returning now to the general discussion of sample preparation, crude DNA is often separated from the cellular debris by a centrifugation step. Prior to hybridization, double-stranded DNA is denatured into single-stranded form. Denaturation of the double-stranded DNA has generally been performed by the techniques involving heating ( greater than Tm), changing salt concentration, addition of base (e.g., NaOH), or denaturing reagents (e.g., urea, formamide). Workers have suggested denaturing DNA into its single-stranded form in an electrochemical cell. The theory is stated to be that there is electron transfer to the DNA at the interface of an electrode, which effectively weakens the double-stranded structure and results in separation of the strands. See, e.g., Stanley, xe2x80x9cDNA Denaturation by an Electric Potentialxe2x80x9d, U.K. patent application 2,247,889 published Mar. 18, 1992.
Nucleic acid hybridization analysis generally involves the detection of a very small number of specific target nucleic acids (DNA or RNA) with an excess of probe DNA, among a relatively large amount of complex non-target nucleic acids. DNA complexity is sometimes overcome to some degree by amplification of target nucleic acid sequences using polymerase chain reaction (PCR). (See, M. A. Innis et al, PCR Protocols: A Guide to Methods and Applications, Academic Press, 1990). While amplification results in an enormous number of target nucleic acid sequences that improves the subsequent direct probe hybridization step, amplification involves lengthy and cumbersome procedures that typically must be performed on a stand alone basis relative to the other substeps. Complicated and relatively large equipment is required to perform the amplification step.
The actual hybridization reaction represents an important step and occurs near the end of the process. The hybridization step involves exposing the prepared DNA sample to a specific reporter probe, at a set of optimal conditions for hybridization to occur to the target DNA sequence. Hybridization may be performed in any one of a number of formats. For example, multiple sample nucleic acid hybridization analysis can be conducted on a variety of filter and solid support formats (See, G. A. Beltz et al., in Methods in Enzymology, Vol. 100, Part B, R. Wu, L. Grossman, K. Moldave, Eds., Academic Press, New York, Chapter 19, pp. 266-308, 1985). One format, the so-called xe2x80x9cdot blotxe2x80x9d hybridization, involves the non-covalent attachment of target DNAs to a filter, which are subsequently hybridized with a radioisotope labelled probe(s). xe2x80x9cDot blotxe2x80x9d hybridization has gained wide-spread use, and many versions have been developed (See, M. L. M. Anderson and B. D. Young, in Nucleic Acid Hybridizationxe2x80x94A Practical Approach, B. D. Hames and S. J. Higgins, Eds., IRL Press, Washington, D.C. Chapter 4, pp. 73-111, 1985). xe2x80x9cDot blotxe2x80x9d assays have been developed for the multiple analysis of genomic mutations (D. Nanibhushan and D. Rabin, in EPA 0228075, Jul. 8, 1987) and for the detection of overlapping clones and the construction of genomic maps (G. A. Evans, in U.S. Pat. No. 5,219,726, Jun. 15, 1993).
New techniques are being developed for carrying out multiple sample nucleic acid hybridization analysis on micro-formatted multiplex or matrix devices (e.g., DNA chips) (See, M. Barinaga, 253 Science, pp. 1489, 1991; W. Bains, 10 Bio/Technology, pp. 757-758, 1992). These methods usually attach specific DNA sequences to very small specific areas of a solid support, such as micro-wells of a DNA chip. These hybridization formats are micro-scale versions of the conventional xe2x80x9cdot blotxe2x80x9d and xe2x80x9csandwichxe2x80x9d hybridization systems.
The micro-formatted hybridization can be used to carry out xe2x80x9csequencing by hybridizationxe2x80x9d (SBH) (See, M. Barinaga, 253 Science, pp. 1489, 1991; W. Bains, 10 Bio/Technology, pp. 757-758, 1992). SBH makes use of all possible n-nucleotide oligomers (n-mers) to identify n-mers in an unknown DNA sample, which are subsequently aligned by algorithm analysis to produce the DNA sequence (R. Drmanac and R. Crkvenjakov, Yugoslav Patent Application #570/87, 1987; R. Drmanac et al., 4 Genomics, 114, 1989; Strezoska et al., 88 Proc. Natl. Acad. Sci. USA 10089, 1992; and R. Dramanac and R. B. Crkvenjakov, U.S. Pat. No. 5,202,231, Apr. 13, 1993).
There are two formats for carrying out SBH. The first format involves creating an array of all possible n-mers on a support, which is then hybridized with the target sequence. The second format involves attaching the target sequence to a support, which is sequentially probed with all possible n-mers. Both formats have the fundamental problems of direct probe hybridizations and additional difficulties related to multiplex hybridizations.
Southern, United Kingdom Patent Application GB 8810400, 1988; E. M. Southern et al., 13 Genomics 1008, 1992, proposed using the first format to analyze or sequence DNA. Southern identified a known single point mutation using PCR amplified genomic DNA. Southern also described a method for synthesizing an array of oligonucleotides on a solid support for SBH. However, Southern did not address how to achieve an optimal stringency condition for each oligonucleotide on an array.
Concurrently, Drmanac et al., 260 Science 1649-1652, 1993, used the second format to sequence several short (116 bp) DNA sequences. Target DNAs were attached to membrane supports (xe2x80x9cdot blotxe2x80x9d format). Each filter was sequentially hybridized with 272 labelled 10-mer and 11-mer oligonucleotides. A wide range of stringency conditions was used to achieve specific hybridization for each n-mer probe; washing times varied from 5 minutes to overnight, and temperatures from 0xc2x0 C. to 16xc2x0 C. Most probes required 3 hours of washing at 16xc2x0 C. The filters had to be exposed for 2 to 18 hours in order to detect hybridization signals. The overall false positive hybridization rate was 5% in spite of the simple target sequences, the reduced set of oligomer probes, and the use of the most stringent conditions available.
A variety of methods exist for detection and analysis of hybridization events. Depending on the reporter group (fluorophore, enzyme, radioisotope, etc.) used to label the DNA probe, detection and analysis are carried out fluorometrically, colorimetrically, or by autoradiography. By observing and measuring emitted radiation, such as fluorescent radiation or particle emission, information may be obtained about the hybridization events. Even when detection methods have very Cr high intrinsic sensitivity, detection of hybridization events is difficult because of the background presence of non-specifically bound materials. A number of other factors also reduce the sensitivity and selectivity of DNA hybridization assays.
Attempts have been made to combine certain processing steps or substeps together. For example, various microrobotic systems have been proposed for preparing arrays of DNA probes on a support material. For example, Beattie et al., in The 1992 San Diego Conference: Genetic Recognition, November, 1992, used a microrobotic system to deposit micro-droplets containing specific DNA sequences into individual microfabricated sample Wells on a glass substrate. Various attempts have been made to describe integrated systems formed on a single chip or substrate, wherein multiple steps of an overall sample preparation and diagnostic system would be included. For example, A. Manz et al., in xe2x80x9cMiniaturized Total Chemical Analysis System: A Novel Concept For Chemical Sensingxe2x80x9d, Sensors And Actuators, B1(1990), pp. 244-248, describe a xe2x80x98total chemical analysis systemxe2x80x99 (TAS) which comprises a modular construction of a miniaturized total chemical analysis system. Sampling, sample transport, any necessary chemical reactions, chromatographic separations as well as detection were to be automatically carried out. Yet another proposed integrated system is Stapleton, U.S. Pat. No. 5,451,500, which describes a system for the automated detection of target nucleic acid sequences in which multiple biological samples are individually incorporated into matrices containing carriers in a 2-dimensional format. Different types of carriers are described for different kinds of diagnostic tests or test panels.
Various multiple electrode systems are disclosed which purport to perform multiple aspects of biological sample preparation or analysis. Pace, U.S. Pat. No. 4,908,112, entitled xe2x80x9cSilicon Semiconductor Wafer for Analyzing Micronic Biological Samplesxe2x80x9d describes an analytical separation device in which a capillary-sized conduit is formed by a channel in a semiconductor device, wherein electrodes are positioned in the channel to activate motion of liquids through the conduit. Pace states that the dimension transverse to the conduit is less than 100 xcexcm. Pace states that all functions of an analytical instrument may be integrated within a single silicon wafer: sample injection, reagent introduction, purification, detection, signal conditioning circuitry, logic and on-board intelligence. Soane et al., in U.S. Pat. No. 5,126,022, entitled xe2x80x9cMethod and Device for Moving Molecules by the Application of a Plurality of Electrical Fieldsxe2x80x9d, describes a system by which materials are moved through trenches by application of electric potentials to electrodes in which selected components may be guided to various trenches filled with antigen-antibodies reactive with given charged particles being moved in the medium or moved into contact with complementary components, dyes, fluorescent tags, radiolabels, enzyme-specific tags or other types of chemicals for any number of purposes such as various transformations which are either physical or chemical in nature. It is said that bacterial or mammalian cells, or viruses may be sorted by complicated trench networks by application of potentials to electrodes where movement through the trench network of the cells or viruses by application of the fields is based upon the size, charge or shape of the particular material being moved. Clark, U.S. Pat. No. 5,194,133, entitled xe2x80x9cSensor Devicesxe2x80x9d, discloses a sensor device for the analysis of a sample fluid which includes a substrate in a surface of which is formed an elongate micro-machined channel containing a material, such as starch, agarose, alginate, carrageenan or polyacrylamide polymer gel, for causing separation of the sample fluid as the fluid passes along the channel. The biological material may comprise, for example, a binding protein, an antibody, a lectin, an enzyme, a sequence of enzymes or a lipid.
Various devices for eluting DNA from various surfaces are known. Shukla U.S. Pat. No. 5,340,449, entitled xe2x80x9cApparatus for Electroelutionxe2x80x9d describes a system and method for the elution of macromolecules such as proteins, DNA and RNA from solid phase matrix materials such as polyacrylamide, agarose and membranes such as PVDF in an electric field. Materials are eluted from the solid phase into a volume defined in part by molecular weight cut-off membranes. Okano, U.S. Pat. No. 5,434,049, entitled xe2x80x9cSeparation of Polynucleotides Using Supports Having a Plurality of Electrode-Containing Cellsxe2x80x9d discloses a method for detecting a plurality of target polynucleotides in a sample, the method including the step of applying a potential to individual chambers so as to serve as electrodes to elute captured target polynucleotides, the eluted material then available for collection.
Generally, the prior art processes have been extremely labor and time intensive. For example, the PCR amplification process is time consuming and adds cost to the diagnostic assay. Multiple steps requiring human intervention either during the process or between processes is suboptimal in that there is a possibility of contamination and operator error. Further, the use of multiple machines or complicated robotic systems for performing the individual processes is often prohibitive except for the largest laboratories, both in terms of the expense and physical space requirements.
As is apparent from the preceding discussion, numerous attempts have been made to provide effective techniques to conduct sample preparation reactions. However, for the reasons stated above, these techniques are limited and lacking. These various approaches are not easily combined to form a system which can carry out a complete DNA diagnostic assay. Despite the long-recognized need for such a system, no satisfactory solution has been proposed previously.
This invention relates broadly to the methods and apparatus for electronic sample preparation of biological materials for their ultimate use in diagnosis or analysis. An integrated system is provided for performing some or all of the processes of: receipt of biological materials, cell selection, sample purification, complexity reduction and/or diagnosis and analysis. Separation of desired components, such as DNA, RNA or proteins from crude mixtures such as biological materials or cell lysates. Electronic sample preparation utilizes the differential mobility and/or differential affinity for various materials in the sample for purposes of preparation and separation. These methods and apparatus are especially useful for the free field electrophoretic purification of DNA from a crude mixture or lysate. In one aspect of this invention, a device comprises at least a first central or sample chamber adapted to receive a buffer solution and a second central or sample chamber adapted to receive a same or different buffer solution, where the first sample chamber and the second sample chamber are separated by a spacer region which preferably includes a trap, membrane or other affinity material. A first electrode in electrical contact with the conductive solution of the first sample chamber and a second electrode in electrical contact with the conductive solution of the second sample chamber are provided. Preferably, each electrode is contained within its own electrode chamber, the electrode chamber being separated from the corresponding sample chamber via a protective layer or separation medium, e.g., a membrane, such as an ultrafiltration membrane, a polymer or a gel. In operation, the sample mixture is placed within the first central chamber. The central chambers contain a solution, preferably a low conductivity buffer solution, such as 50 mM histidine, 250 mM HEPES, or 0.5xc3x97TBE. A mixture of biological substances, for example a crude lysate, is added to the first chamber and then the electrodes are activated. Substances with mobility in an electric field will move towards one electrode or the other depending on the charge of the substance. In one embodiment, the desired and undesired substances with similar charges will be attracted to an electrode biased with the opposite charge to the desired substance, located in the second chamber. Therefore, the desired substance and the undesired substances of similar charge will move towards the affinity material which is between the first and second chambers.
In one embodiment, the sample mixture is composed of a desired substance which has charge mixed with undesired substances some of which have charges. After the electrodes are activated, the desired substance travels toward the second chamber where the electrode, biased with opposite charge, is located and binds to the affinity material. In contrast, the similarly charged, undesired substances move toward the electrode biased with the opposite charge and pass through the affinity material into the second chamber. Other undesired substances with the opposite charge will be attracted toward the electrode in the first chamber. After the undesired substances have passed through the affinity material, the electrolyte solution may be changed in both chambers to remove the undesired substances. Then the desired substance is eluted into the fresh electrolyte solution. Elution can be accomplished by continued electrophoresis at the same or increased current or by the addition of a chemical, such as a detergent, salt, a base or an acid, that will cause elution from the affinity material. In addition, a change in temperature could be used to elute the desired substance.
In one particular embodiment, the affinity material is composed of a gel with sufficient volume to hold a substantial fraction, e.g., preferably 50%, and more preferably, 80%, of the desired substance in the sample. The gel composition and concentration is chosen such that the mobility of the desired substance which has high molecular weight (30,000 to 3,000,000 daltons) is retarded by the gel but the mobility of the undesired substances which have low molecular weight (100 to 10,000 daltons) is relatively unaffected. Consequently, the gel will release the desired substance only after a longer period of electrophoresis or a higher current than is necessary for the passage of the undesired substances. In effect the gel is a trap for the desired substance but does not provide a relatively significant barrier to undesired substances. The desired and undesired substances preferably have a very large difference in electrophoretic mobility while traveling through the gel for the gel to serve as a trap. Preferably, the trap does not effectively resolve mobility differences among fragments which have similar compositions and molecular weights which are within a factor of substantially 10 of each other.
In accordance with this invention, the resolution of different molecular weights is preferentially sacrificed in favor of rapid mobility. Preferably, the gel in the device is relatively compact (e.g., 0.5 to 10 mm) in the direction of migration to permit rapid electrophoretic transport. Thus, the speed of this technique is not compatible with conventional resolution of substances by size except between substances with very gross differences in size. Consequently, it is inherent in this technique that desired substances of very different molecular weights will be copurified. The preparation of substances of similar composition but different sizes may be advantageous to the user as for example in the purification of DNA of different sizes for the purpose of cloning of a whole or representative portion of a genome of an organism.
In accordance with one aspect of this invention, the desired substance is propelled into and out of the gel in the same device. That is, in the preferred embodiment, different regions within the whole system serve as traps and therefore, help to separate analytes or materials to be eluted. The integration of electrophoresis and elution steps also provides significant advantages for the user in saving time, reducing the number of steps required and decreasing the amount of space required for the apparatus.
In one aspect of the invention, a device containing multiple electrode chambers in electrical communication with a first and second end sample chamber and one or more intermediate sample chambers is advantageously utilized. In the preferred embodiment, each of the end sample chambers and the intermediate sample chamber or chambers is in electrical communication with an electrode chamber, preferably having the sample chamber separated from the electrode chamber by a membrane. Preferably, the electrode chamber has a buffer volume which is larger than and preferably much larger than (e.g., at least 10 to 1) the volume of the sample which is loaded into the sample chamber.
In another aspect of this invention, the traps, membranes or other affinity materials serve as a transporter or xe2x80x98dipstickxe2x80x99 to collect and permit transport of materials. In one embodiment of this device, the membrane or other affinity material disposed between adjacent sample chambers is provided with structural integrity to permit the removal of the trap or affinity material from the chamber structures. In one preferred embodiment, a membrane or trap is held in a frame which is adapted to mate with a channel formed at the spacer region. The transporter or dipstick may be utilized to transport the collected material for further processing, such as further purification, complexity reduction or assaying or diagnosis. Optionally, the transporter may be disposed adjacent to or formed in electrical communication with a power source.
In yet another aspect of this invention, first and second electrodes are disposed within an intervening sample solution, the system further including a third electrode between the first and second electrodes. The third electrode may serve as a control electrode to modulate the flow of charged materials within the sample solution. The third electrode is preferably formed as a grid, and may be advantageously formed by sputtering a metal coating on a membrane. The third electrode, or grid, is preferably disposed within the sample solution closer to the first electrode than the second electrode. In one mode of operation, a sample is placed within the sample solution between the first and third electrodes, and the first and second electrodes are biased for net migration of charged materials of a first charge toward the second electrode. The third electrode or grid is preferably biased slightly negative or neutral. Once the desired DNA or other charged materials passes through or by the third electrode or grid, the third electrode or grid is preferably made relatively negative. This increased negativity serves to move the negatively charged DNA towards the second electrode, and to repel other, more slowly moving negatively charged materials which still remain between the first electrode and the third or grid electrode.
In yet another aspect of this invention, a pair of electrodes are located within a sample solution and are operated so as to bunch or concentrate a subset of the charged macromolecules within the sample solution. Biasing one electrode so as to accelerate motion of charged macromolecules towards the other electrode, and biasing the other electrode so as to retard motion of the charged macromolecules which are closer to that electrode in the region between the electrodes. Such bunching serves to physically concentrate the charged macromolecules within the region.
In a preferred embodiment, a xe2x80x9cCxe2x80x9d shaped electrode is utilized. This structure serves to bunch materials contained within the region bounded by the C-shaped electrode, as well as to repel like charged materials which are external to the region bounded by the C-shaped region. Further, the materials contained within the C-shaped region are subject to a force in a sideways (i.e., a transverse or oblique direction to the net flow direction apart from the C-shaped electrode). The C-shaped electrode may be an integrated, continuous electrode or may be segmented. Other shapes are advantageously utilized, such as parabolic structures. The C-shaped region is sized to include within the region the desired or target material.
A complexity reduction device includes one or more probe areas which comprise a support material, preferably a polymer gel, such as an agarose, acrylamide or other conductive polymer, to which capture probes are attached. The support material is formed in contact with an electrode to permit the electrophoretic attraction and hybridization of the capture probes with the target materials. Electrical elution or electronic stringency control of the captured materials may be used. In one embodiment, the complexity reduction device is formed from the combination of a chamber mated to a printed circuit board. The chamber includes vias in which the support material is located. The printed circuit board preferably including concentric vias to provide a continuous space for the inclusion of the support material. In this embodiment, gases or other reaction products may be vented through the via, in part because the gas generally does not rise into the via since it is filled with polymer. Therefore, these gases or other reaction products do not come in contact with the analytes, such as DNA. The complexity reduction system optionally may further include disposal regions for the attraction and/or disposal of undesired materials. The disposal regions include an electrode in electrical communication through the conductive polymer. In operation, the complexity reduction device performs free field electrophoretic transport. Alternatively, an uncovered electrode in contact with the solution may be utilized for disposal of undesired materials.
In yet another aspect of this invention, a DNA or other nucleic acid purification device is provided. An upper reservoir containing an electrode, which may be identified as a cathode, is adapted to receive a buffer solution and a sample solution. Preferably, the upstream reservoir includes a tube in fluid communication with the upstream reservoir, the tube having an internal diameter less than the diameter of the upstream reservoir. The tube includes at least a first differential mobility section, preferably a gel, which provides a plug or trap region within the tube. Optionally, the gel may be cast on top of a support membrane. A collection chamber is adjacent to the differential mobility region. In the preferred embodiment, the collection region has a smaller volume than the differential mobility region and is smaller than the sample volume. The reduction in volume from sample to collection permits an increase in volume concentration of DNA and exchanges the DNA into the desired buffer formulation of known volume. An anode is provided in a lower reservoir. In yet another aspect of this invention, the format and transport of DNA is in the horizontal plane instead of vertical.
In operation, a sample is subject to a cell lysing and shearing pre-preparation step. Preferably, the proteins are then reduced in size, such as through application of a proteinase, such as proteinase K. Preferably, when the unit is operated in a vertical format, sucrose or other densifier is added to the sample. The densifier serves to collect and concentrate the sample in a region immediately above the first differential mobility section. The prepared sample including densifier is then injected onto the separation unit in the tube, in a region immediately upstream of the differential mobility region. The cathode and anode are then energized to provide electrophoretic transport of the charged materials within the system, causing the reduced size protein materials to pass through the differential separation medium first, while retaining the relatively slower moving DNA, as well as resulting in the proteinase K or other positively charged materials being removed to the cathode. After a time sufficient to permit desired amounts of protein to pass through the differential mobility region and support membrane, the DNA is eluted from the differential movement region into the sample chamber. Optionally, the buffer solution in the lower reservoir which received the proteinaceous material having passed through the support membrane may be removed and replaced with new buffer solution, and optionally provided with a membrane which serves to retain eluted DNA within the sample chamber.
In yet another aspect of this invention, the cathode and anode electrodes are in communication with the sample through conductive fluids or gel or polymers, but are resident in the power supply or other controlling instrumentation. Conduction is made through fluidic ports and the electrode (noble metal) preferably is not a part of the consumable device.
Integrated systems may be advantageously formed which include some or all of the functions of cell separation, purification, complexity reduction and diagnostics. In one embodiment, a purification chamber includes an input port and multiple electrodes for providing an electrophoretic driving force to the charged materials. A protein trap disposed between the sample input port and an outlet tap serves to trap undesired proteins or other charged macromolecules. In an alternative embodiment, the undesired materials such as proteins are reduced in size or modified in charge so as to increase their degree of mobility relative to the desired materials, such as DNA. The materials for further processing are then removed from the trap or other transportation device for further processing, such as complexity reduction and/or diagnostic procedures. This separation of target materials from the undesired material is accomplished by modifying the electric field to steer the target materials into a region of higher purity (e.g., pure buffer). Electric field modification can be implemented through C-shape electrodes or activation of a new configuration of electrodes which favorably influence the direction of mobility of the target.
In yet another aspect of this invention, an integrated sample preparation, complexity reduction and diagnostic system is provided. An input port receives crude lysate including proteins which have been reduced in size, such as through use of a proteinase. The materials pass through a DNA trap, in which the DNA moves relatively slower than the proteins. The proteins arrive at a protein trap electrophoretically prior to the arrival of the DNA. The DNA then exit the DNA trap towards a collections chamber. Preferably, the collection chamber has a volume which is less than, preferably substantially less than the volume of the DNA trap, such as 50 microliters. This volume is in communication with the complexity reduction and diagnostic chamber. In one aspect of this invention, fluidic transport within the device is accomplished by input ports located at each end of the collection chamber. These dual input ports are adapted to receive any fluid, such as a buffer, an air slug or a reagent. By selective operation of supplies or pumps, the materials contained within the collection chamber e.g., substantially purified DNA, may be forced from the chamber to the complexity reduction device. By utilization of air slugs, various liquids may be separated from one another. By operation of the input liquids or gas, a hydraulic or pneumatic ram serves to move materials within the fluid section of the device. While materials may be pushed forward throughout the system, such as from the collection volume to the complexity reduction to the diagnostic region, the materials may be moved in the opposite direction, e.g., from the complexity reduction chamber to the concentration region.
Accordingly, it is an object of this invention to provide for a sample preparation system useful for biological sample preparation.
It is yet a further object of this invention to provide a system for purification of DNA from biological materials or other crude samples.
It is yet a further object of this invention to provide methods for the separation and purification of desired biological charged macromolecules through differential solution phase mobility and/or differential affinity.